Log of James Marr for the Chemical Information Retrieval Class at Drexel University (Fall 2012)
All 25 chemical properties have been entered (14-Oct-2012).
Final Research Paper Topic: Utilizing High pH Mobile Phase In LC-MS Experiments In The Pharmaceutical Industry
Assignment #2: (Entered 28-Nov-2012)
Write a summary of one of the articles you are reading for your project paragraph by paragraph. One or two sentences per paragraph is fine. You must do this in your own words. No significant amount of text can be copied from the abstract or any part of the paper. Either put the summary in bullet form on your research log or on AcaWiki. Due Sat Dec 1, 2012 9:00AM
Authors: Jean-Nicholas Mess, Mathieu Lahaie, Milton Furtado, and Fabio Garofolo
Abstract
Using post column addition of high pH modifiers, 25 compounds were seen to have either no change or enhancement in signal when using LC-ESI+-MS/MS; allowing for the group to do a quick sample preparation of candesartan not normally done under low pH conditions.
Introduction
Using a high pH mobile phase can help overcome the issues seen when running basic drugs under acidic or neutral pH HPLC conditions.
A high pH mobile phase will deprotonate the basic analytes, causing the analyte to elute under higher organic conditions, away from the problematic solvent front.
Despite the fact that ESI detection relies on the charge state of the analyte in solution, a high pH mobile phase still allows the analyte to have adequate sensitivity.
The just as good or better limits of detection under high pH conditions, compared to acidic conditions, could potentially come from the LC rather than the MS side due to the fact that the analytes eluted with higher organic and better peak shape.
This study looked at the impact of adding the high pH modifiers by postcolumn addition, allowing for no chromatographic differences to come from the LC. The data suggests that sensitivity is increased under positive ESI conditions, allowing a quick bioassay to be developed.
Experiment
Formic acid, acetic acid, ammonium acetate, ammonium bicarbonate, ammonium hydroxide, methanol, and water were used.
Reference standards were purchased from Pharmacopia, Synfine Research, Richland Chemicals, Jinglong Pharmatech, and Cerilliant.
One Agilent 1100 LC system was used for chromatographic separation and one Agilent 1100 LC system was used for postcolumn addition of the modifiers. An API300 with TIS was used for detection.
5uL of one solution containing all compounds was injected on to an XBridge Phenyl 30x4.6mm, 3.5um column with a gradient elution of 2.5min (total run time of 5.5min) at 35 deg C. Modifiers, all prepared in water, were added at 200uL/min post column via a T-union.
Using ESI, [M+H]+ ion was monitored for all compounds on an API3000 with all initial instrument parameters maintained throughout the experiment.
Samples of Candesartan were prepared via protein precipitation and injected under both acidic and high pH conditions.
Results and Discussion
Using an API3000 with an ESI source in positive mode, the post-column addition of high pH was studied on a mixture of 25 analytes.
Adding the high pH post column ensured that the high pH did not impact the chromatography of the mixture. Running in this manner allows for the assumption to be made that any changes in analyte sensitivity is due to the high pH modifiers added rather than changes in chromatography.
When comparing ammonium hydroxide to formic acid, almost half of the analytes increased in sensitivity. When comparing ammonium hydroxide to acetic acid, all but two compounds had an increase in sensitivity, with some having >8 fold increase in sensitivity. These results agree with others who allowed chromatographic parameters to be altered by the pH of the mobile phase.
In testing a mobile phase at pH 6.0 (ammonium acetate adjusted with acetic acid) against one at pH 9.3 (ammonium acetate adjusted with ammonium hydroxide), the data generated from the higher pH mobile phase showed more sensitivty, allowing one to come to the conclusion that just increasing the pH of the mobile phase will increase the signal in most cases.
Another test compared mobile phase with ammonium bicarbonate adjusted to pH 7.8 and 10.5 and one with ammonium acetate adjusted to pH 9.3. The results from this test showed that the analytes run in a mobile phase with ammonium bicarbonate, no matter what pH it was adjusted to, were more sensitive than when run in the ammonium acetate mobile phase. Interestingly, there was no sensitivity difference seen between the analytes run in the different pH ammonium bicarbonate mobile phases, allowing one to draw the conclusion that the mobile phase buffer also plays a role in the sensitivity gain.
Results from high pH vs. low pH studies conducted by others are similar in nature to the findings from the authors of this paper.
"Wrong-way-round" ionization is not completely understood, but there are three theories that could be used to explain the results obtained in this paper, the first of which is gas phase ion-molecule reactions between the analyte and ammonium ion.
Another theory involves collision induced dissociation of an analyte-ammonium ion cluster.
The third theory involves a proton transfer in solution from the ammonium ion to the analyte as ammonia evaporates off.
These theories can explain why ions are produced in ESI+ even though the analyte is neutral in solution, but they cannot explain why this is the case under high pH. It may be because the hydronium ion in acidic condtions is not as good of a proton donor as the ammoium ion in basic conditions.
Due to what was found out in the experiments run, a bioanalytical method for candesartan, using a protein precipitation extraction of human plasma, was developed to reach a lower limit of quantitation of 1.00 ng/mL. Running under high pH conditions, candesartan eluted at a similar retention as low pH conditions, but had a 4 fold increase in sensitivity.
Columns designed to run under high pH conditions allow for analytes to be separated in their unionized state and receive the benefits of later elution. The study run with candesartan shows that the advantages achieved by running under high pH conditions on a high pH stable column are also achieved for acidic analytes.
One must always exercise caution, even under high pH conditions, when developing a method. In this particular case, candesartan did not remain at a constant retention time when ammonium hydroxide was used, but did when ammonium bicarbonate was used to buffer the mobile phase, suggesting that buffered mobile phases may be able to give more reproducible chromatograms.
Conclusion
High pH modifiers were found to enhance the signal of a majority of tested analytes, including the acidic analyte candesartan, when added post column under ESI+ conditions, adding to the already established chromatographic advantages of high pH for basic analytes. The enhanced sensitivty gained from the high pH environment allows for a fast protein precipitation extraction method to be utilized when previously, under acidic mobile phase conditions, the samples were not clean enough to achieve the desired LLOQ.
Future Perspective
The types of columns stable at high pH are not as vast as those stable at low pH, but with continued research in to synthesis of column stationary phases, more columns will become available. This will allow for high pH chromatography to be just as popular as neutral and low pH chromatography are now.
Ethical Conduct of Research
Appropriate guidelines were followed and approval had been acquired for all human and animal experiments.
Financial & Competing Interest Disclosure
The authors had no association, financial or otherwise, with any organization having a financial interest or conflict with what was discussed in the paper.
All 25 chemical properties have been entered (14-Oct-2012).
Final Research Paper Topic: Utilizing High pH Mobile Phase In LC-MS Experiments In The Pharmaceutical Industry
Assignment #2: (Entered 28-Nov-2012)
Write a summary of one of the articles you are reading for your project paragraph by paragraph. One or two sentences per paragraph is fine. You must do this in your own words. No significant amount of text can be copied from the abstract or any part of the paper. Either put the summary in bullet form on your research log or on AcaWiki. Due Sat Dec 1, 2012 9:00AM
Article: Effect of high pH mobile phase on the sensitivity of multiple drugs by LC positive electrospray ionization MS/MS (linked to article)
Authors: Jean-Nicholas Mess, Mathieu Lahaie, Milton Furtado, and Fabio Garofolo
Abstract
- Using post column addition of high pH modifiers, 25 compounds were seen to have either no change or enhancement in signal when using LC-ESI+-MS/MS; allowing for the group to do a quick sample preparation of candesartan not normally done under low pH conditions.
Introduction- Using a high pH mobile phase can help overcome the issues seen when running basic drugs under acidic or neutral pH HPLC conditions.
- A high pH mobile phase will deprotonate the basic analytes, causing the analyte to elute under higher organic conditions, away from the problematic solvent front.
- Despite the fact that ESI detection relies on the charge state of the analyte in solution, a high pH mobile phase still allows the analyte to have adequate sensitivity.
- The just as good or better limits of detection under high pH conditions, compared to acidic conditions, could potentially come from the LC rather than the MS side due to the fact that the analytes eluted with higher organic and better peak shape.
- This study looked at the impact of adding the high pH modifiers by postcolumn addition, allowing for no chromatographic differences to come from the LC. The data suggests that sensitivity is increased under positive ESI conditions, allowing a quick bioassay to be developed.
Experiment- Formic acid, acetic acid, ammonium acetate, ammonium bicarbonate, ammonium hydroxide, methanol, and water were used.
- Reference standards were purchased from Pharmacopia, Synfine Research, Richland Chemicals, Jinglong Pharmatech, and Cerilliant.
- One Agilent 1100 LC system was used for chromatographic separation and one Agilent 1100 LC system was used for postcolumn addition of the modifiers. An API300 with TIS was used for detection.
- 5uL of one solution containing all compounds was injected on to an XBridge Phenyl 30x4.6mm, 3.5um column with a gradient elution of 2.5min (total run time of 5.5min) at 35 deg C. Modifiers, all prepared in water, were added at 200uL/min post column via a T-union.
- Using ESI, [M+H]+ ion was monitored for all compounds on an API3000 with all initial instrument parameters maintained throughout the experiment.
- Samples of Candesartan were prepared via protein precipitation and injected under both acidic and high pH conditions.
Results and Discussion- Using an API3000 with an ESI source in positive mode, the post-column addition of high pH was studied on a mixture of 25 analytes.
- Adding the high pH post column ensured that the high pH did not impact the chromatography of the mixture. Running in this manner allows for the assumption to be made that any changes in analyte sensitivity is due to the high pH modifiers added rather than changes in chromatography.
- When comparing ammonium hydroxide to formic acid, almost half of the analytes increased in sensitivity. When comparing ammonium hydroxide to acetic acid, all but two compounds had an increase in sensitivity, with some having >8 fold increase in sensitivity. These results agree with others who allowed chromatographic parameters to be altered by the pH of the mobile phase.
- In testing a mobile phase at pH 6.0 (ammonium acetate adjusted with acetic acid) against one at pH 9.3 (ammonium acetate adjusted with ammonium hydroxide), the data generated from the higher pH mobile phase showed more sensitivty, allowing one to come to the conclusion that just increasing the pH of the mobile phase will increase the signal in most cases.
- Another test compared mobile phase with ammonium bicarbonate adjusted to pH 7.8 and 10.5 and one with ammonium acetate adjusted to pH 9.3. The results from this test showed that the analytes run in a mobile phase with ammonium bicarbonate, no matter what pH it was adjusted to, were more sensitive than when run in the ammonium acetate mobile phase. Interestingly, there was no sensitivity difference seen between the analytes run in the different pH ammonium bicarbonate mobile phases, allowing one to draw the conclusion that the mobile phase buffer also plays a role in the sensitivity gain.
- Results from high pH vs. low pH studies conducted by others are similar in nature to the findings from the authors of this paper.
- "Wrong-way-round" ionization is not completely understood, but there are three theories that could be used to explain the results obtained in this paper, the first of which is gas phase ion-molecule reactions between the analyte and ammonium ion.
- Another theory involves collision induced dissociation of an analyte-ammonium ion cluster.
- The third theory involves a proton transfer in solution from the ammonium ion to the analyte as ammonia evaporates off.
- These theories can explain why ions are produced in ESI+ even though the analyte is neutral in solution, but they cannot explain why this is the case under high pH. It may be because the hydronium ion in acidic condtions is not as good of a proton donor as the ammoium ion in basic conditions.
- Due to what was found out in the experiments run, a bioanalytical method for candesartan, using a protein precipitation extraction of human plasma, was developed to reach a lower limit of quantitation of 1.00 ng/mL. Running under high pH conditions, candesartan eluted at a similar retention as low pH conditions, but had a 4 fold increase in sensitivity.
- Columns designed to run under high pH conditions allow for analytes to be separated in their unionized state and receive the benefits of later elution. The study run with candesartan shows that the advantages achieved by running under high pH conditions on a high pH stable column are also achieved for acidic analytes.
- One must always exercise caution, even under high pH conditions, when developing a method. In this particular case, candesartan did not remain at a constant retention time when ammonium hydroxide was used, but did when ammonium bicarbonate was used to buffer the mobile phase, suggesting that buffered mobile phases may be able to give more reproducible chromatograms.
Conclusion- High pH modifiers were found to enhance the signal of a majority of tested analytes, including the acidic analyte candesartan, when added post column under ESI+ conditions, adding to the already established chromatographic advantages of high pH for basic analytes. The enhanced sensitivty gained from the high pH environment allows for a fast protein precipitation extraction method to be utilized when previously, under acidic mobile phase conditions, the samples were not clean enough to achieve the desired LLOQ.
Future Perspective- The types of columns stable at high pH are not as vast as those stable at low pH, but with continued research in to synthesis of column stationary phases, more columns will become available. This will allow for high pH chromatography to be just as popular as neutral and low pH chromatography are now.
Ethical Conduct of Research- Appropriate guidelines were followed and approval had been acquired for all human and animal experiments.
Financial & Competing Interest Disclosure- The authors had no association, financial or otherwise, with any organization having a financial interest or conflict with what was discussed in the paper.
- No assistance was used writing the paper.
End of Paper